How to fix peak splitting on acetate peak

[Merachroma]

Hello,

I am a student starting my internship investigating wood samples that are deacetylated in which I collect acetate. This acetate mixture is what I measure on the HPLC but I have peak splitting. As wood is a natural product there are a lot of extractives present but I am solely interested in the acetate peak and the IS butyrate.

I am not sure if it is peak splitting but it could also be a component with the same retention but I am more sure of it being a shoulder peak.

My column is a 100 x 7.7 mm H+ packed with sulfonated styrene /divinylbenzene copolymer.
- Detector: UV at 205 nm
- Mobile phase: MilliQ/H2SO4 (1000ml:0.5ml)
no gradient.

- Oven: 40 degrees for first 8 minutes
- Flow: 0.8 ml/min

In the picture below I have shown my chromatogram with the red arrow indicating the current problem.

Any ideas are welcome.

[millerk8561]

My first gut check would be to say, do you have an acetate standard you can run for comparison purposes?

[anionman]

Run formate too. in IC columns formate follows acetate closely. We get a lot coming out of the plastic vials and it can interfere with our fluoride analysis. If a sample is heavily chlorinated it leaches a lot of formate and acetate out of the plastic and can make fluoride analysis impossible. One time I was able to do it by calibrating and quantifying fluoride by peak height instead of area.

It's possible you have a contaminant too. Try reversing the column and /or washing with acetonitrile.

[Merachroma]

My first gut check would be to say, do you have an acetate standard you can run for comparison purposes?

Yep I do. I have multiple actually. A calibration series and two references that both give a regular acetate peak. This problem seems to be specifically for this wood species.

[Merachroma]

Run formate too. in IC columns formate follows acetate closely. We get a lot coming out of the plastic vials and it can interfere with our fluoride analysis. If a sample is heavily chlorinated it leaches a lot of formate and acetate out of the plastic and can make fluoride analysis impossible. One time I was able to do it by calibrating and quantifying fluoride by peak height instead of area.

It's possible you have a contaminant too. Try reversing the column and /or washing with acetonitrile.

Didn't know this was possible, the more you know. My colleague had already ran a formic acid (so formate) test and it gave a complete different retention (it eluded earlier) so I do not think this is caused by formate. This chromatogram is shown below, the black peak at retention time 3.9 is the formate.

Also we did wash the column just last week so that is fine and it's sure that this is an issue for this wood species particularly.

[vmu]

A calibration series and two references that both give a regular acetate peak.

This means that the co-eluted unknown compound in the sample is the most plausible explanation. Try to reduce (2x) and increase (2x) the injection volume and see whether the peak shape changes. Also add some acetate (2x) to the sample and see whether the shoulder increases or remains the same.

[Merachroma]

A calibration series and two references that both give a regular acetate peak.

This means that the co-eluted unknown compound in the sample is the most plausible explanation. Try to reduce (2x) and increase (2x) the injection volume and see whether the peak shape changes. Also add some acetate (2x) to the sample and see whether the shoulder increases or remains the same.

I will try and see if this has made any changes, thank you.